1. Field of the Invention
This invention relates in general to the analysis of blood samples using a particle analyzer, and more particularly to determining reticulocytes.
2. Background
The predominant cell in blood is the erythrocyte, i.e., red blood cell or red cell. In a peripheral blood smear, erythrocytes derive their reddish color from protein hemoglobin, and usually appear round or oval with a pale-staining center region. Their biconcave morphology increases the cell's surface area and facilitates diffusion of oxygen and carbon-dioxide from the cell. A typical erythrocyte has a lifespan of about 120 days.
Erythrocytes develop from nucleated precursor cells in the bone marrow. Immature erythrocytes, i.e., reticulocytes, have organelles that contribute to an increased hemoglobin content and gas-carrying capacity. Reticulocytes may be recognized in peripheral blood smears when a special stain is used to stain their polyribosome or ribonucleic acid (RNA). Under typical conditions, reticulocytes account for about 1-2% of red blood cells in a sample. However, during certain periods of physical need, the reticulocyte count may increase.
The RNA of a reticulocyte degrades and reduces in size over a period of days. Immature reticulocytes are larger and contain a greater quantity of RNA. A measure of reticulocyte maturation can provide an increased understanding of erythrocyte activity.
For example, the immature reticulocyte fraction (IRF), i.e., ratio of immature reticulocytes to total reticulocytes, can be useful in many aspects including evaluating the extent of bone marrow suppression during chemotherapy, monitoring bone marrow engraftment, assessing neonatal transfusion needs, etc.
Automated reticulocyte analysis is done using a particle analyzer such as a flow cytometer or hematology analyzer. Example particle analyzers include, the Gen•S™ System from Beckman Coulter and XT-2000 from Sysmex Corporation. The preparation of a blood sample for flow cytometric or hematology analysis generally involves, taking a whole blood sample and performing one or both the steps of, incubating the sample of blood with a vital stain such as New Methylene Blue (NMB), and diluting the blood sample with a hypotonic acid that clears hemoglobin. The staining precipitates RNA within the erythrocytes. Diluting with a hypotonic acid clears hemoglobin, leaving the stained RNA within the cells. The process of removing hemoglobin is commonly referred to as “ghosting.” The blood sample, or portion of it, is then subjected to analysis in a flow cell of a particle analyzer. Typically, cells in a sheath fluid pass through a point in the flow cell, one by one, where they are interrogated by probes including one or more beams of light. Several measurements are generated for each passing cell. The interrogation of a single cell is referred to as a cell event. Commonly recorded measurements per cell event include, forward light scatter, side scatter, axial light loss, and fluorescence. Some particle analyzers also collect a direct current impedance (DC) measurement which is a measure of how much impedance is exerted by a cell. The DC measurement, which is obtained from applying the maximum current such that the cell membrane is not permeated and no current flows through the cell, is also known as Coulter volume or volume.
Younger erythrocytes have a larger quantity of RNA that cause a higher amount of light to be scattered than from the more mature erythrocytes. For example, light scatter measurements such as side scatter are generally proportional to the number of nucleic granules. Also, the reticulocytes are generally larger than the mature erythrocytes because they have a larger quantity of RNA granules. Other light scatter measurements may also be used to measure the maturity of the cells. For example, a measurement such as forward scatter or fluorescence also indicates the relative amount of RNA in each cell. In addition, the volume of a cell can be directly measured through the DC measurement.
Current reticulocyte analysis methods, in general, use only one measurement indicative of reticulocyte maturity to differentiate reticulocytes. For example, C. Kessler, P. Campbell, V. Bolufe, I. Fernandez, “Immature Reticulocyte Fraction and Reticulocyte Maturity Index”, www.beckmancoulter.com/literature/ClinDiag/recticliterature.pdf (last accessed Jun. 30, 2008), describes a method in which light scatter and volume measurements are used to differentiate reticulocytes from mature red cells, and the light scatter by itself being used for differentiating the reticulocytes according to maturity. As another example, U.S. Patent Publication No. 2004/0132196 describes a method in which fluorescence and forward scatter are used to differentiate reticulocytes from mature red cells, and fluorescence by itself being used for differentiating the reticulocytes according to maturity. The use of a second measurement that is indicative of reticulocyte maturity, may help increase the efficiency and accuracy of reticulocyte analysis.
Therefore, what are needed are methods and systems to more accurately measure reticulocyte populations.